Christine Brostjan

Publications

• Discrimination between circulating endothelial cells and blood cell populations with overlapping phenotype reveals distinct regulation and predictive potential in cancer therapy.

  Patrick Starlinger, Philipp Brugger, Christian Reiter, Dominic Schauer, Silvia Sommerfeldt, Dietmar Tamandl, Irene Kuehrer, Sebastian F Schoppmann, Michael Gnant, Christine Brostjan

  Neoplasia (New York, N.Y.). 10/2011; 13(10):980-90.

  Circulating endothelial cells (CECs) have been proposed to predict patient response to antiangiogenic cancer therapy. However, contradictory reports and inconsistency in the phenotypic identification of CECs have led us to compare three cell populations with partially overlapping phenotype in cancer... [more] Circulating endothelial cells (CECs) have been proposed to predict patient response to antiangiogenic cancer therapy. However, contradictory reports and inconsistency in the phenotypic identification of CECs have led us to compare three cell populations with partially overlapping phenotype in cancer patients receiving chemotherapy and the antiangiogenic agent bevacizumab. Patients (n = 20) with locally advanced pancreatic cancer were monitored during 16 weeks of neoadjuvant treatment with gemcitabine and bevacizumab. Detection of circulating cell populations was based on the marker combination CD45, CD31, and CD146; levels of viable and dead (7-aminoactinomycin D-positive) cells were evaluated by flow cytometry in 2-week intervals. We were able to discriminate and concomitantly monitor three cell populations elevated in cancer patients. Whereas CECs were defined as CD45(-) CD31(+) CD146(+), the distinct populations of CD45(-) CD31(-) CD146(+) and CD45(-) CD31(high) CD146(-) cells were partly positive for CD3 and CD41, respectively. CECs and CD45(-) CD31(-) CD146(+) cells increased during therapy; the rise in dead cells was positively correlated with patient response or survival. Conversely, CD45(-) CD31(high) CD146(-) cells decreased in neoadjuvant treatment. A highly significant correlation was established for improved patient response and a minor decrease in viable cell counts. Flow cytometric CEC analysis based on CD45, CD31, and CD146 requires careful discrimination between blood cell populations with overlapping phenotype showing hallmarks of activated T cells and large platelets. However, these three cell populations show distinct regulation during cancer therapy, and their concomitant analysis may offer extended prognostic and predictive information.

• Myelosuppression of thrombocytes and monocytes is associated with a lack of synergy between chemotherapy and anti-VEGF treatment.

  Patrick Starlinger, Philipp Brugger, Dominic Schauer, Silvia Sommerfeldt, Dietmar Tamandl, Irene Kuehrer, Sebastian F Schoppmann, Michael Gnant, Christine Brostjan

  Neoplasia (New York, N.Y.). 05/2011; 13(5):419-27.

  Chemotherapeutic agents that have shown improved patient outcome when combined with anti-vascular endothelial growth factor (VEGF) therapy were recently identified to induce the mobilization of proangiogenic Tie-2-expressing monocytes (TEMs) and endothelial progenitor cells (EPCs) by platelet releas... [more] Chemotherapeutic agents that have shown improved patient outcome when combined with anti-vascular endothelial growth factor (VEGF) therapy were recently identified to induce the mobilization of proangiogenic Tie-2-expressing monocytes (TEMs) and endothelial progenitor cells (EPCs) by platelet release of stromal cell-derived factor 1α (SDF-1α). VEGF blockade was found to counteract cell mobilization. We aimed to determine why agents like gemcitabine do not elicit TEM and EPC recruitment and may therefore lack synergy with anti-VEGF therapy. Locally advanced pancreatic cancer patients (n = 20) were monitored during 16 weeks of neoadjuvant therapy. Treatment was based on gemcitabine with or without the addition of bevacizumab. Blood levels of proangiogenic cell populations and angiogenesis factors were determined in 2-week intervals. The lack of EPC mobilization during gemcitabine therapy was associated with severe thrombocytopenia and reduced SDF-1α blood concentrations. Furthermore, myelosuppression by gemcitabine correlated significantly with loss of TEMs. With respect to angiogenic factors stored and released by platelets, plasma levels of the angiogenesis inhibitor thrombospondin 1 (TSP-1) were selectively decreased and correlated significantly with thrombocytopenia in response to gemcitabine therapy. A thorough literature screen identified thrombocytopenia as a common feature of chemotherapeutic agents that lack synergy with anti-VEGF treatment. Our results on gemcitabine therapy indicate that myelosuppression (in particular, with respect to thrombocytes and monocytes) interferes with the mobilization of proangiogenic cell types targeted by bevacizumab and may further counteract antiangiogenic therapy by substantially reducing the angiogenesis inhibitor TSP-1.
• 2.41

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  Periodontopathogens induce soluble P-selectin release by endothelial cells and platelets.

  Alice Assinger, Elisabeth Buchberger, Markus Laky, Azadeh Esfandeyari, Christine Brostjan, Ivo Volf

  Thrombosis research. 01/2011; 127(1):e20-6.

  Soluble P-selectin plays a pivotal role in inflammation and the development of thrombotic and cardiovascular disease. Accordingly, elevated levels of soluble P-selectin are found in periodontitis and (other forms of) inflammatory diseases. However, the cellular source of soluble P-selectin in period... [more] Soluble P-selectin plays a pivotal role in inflammation and the development of thrombotic and cardiovascular disease. Accordingly, elevated levels of soluble P-selectin are found in periodontitis and (other forms of) inflammatory diseases. However, the cellular source of soluble P-selectin in periodontitis and the effects of periodontopathogens on P-selectin release are unknown. Soluble P-selectin was determined in 26 patients with periodontitis and 19 controls. Furthermore, human endothelial cells and platelets were investigated for their ability to elicit soluble and surface P-selectin in response to periodontopathogens A. actinomycetemcomitans Y4 and P. gingivalis. Moreover surface E-selectin and ICAM-1 expression as well as NFκB translocation in response to these bacteria were determined on endothelial cells as well as the formation of platelet-leukocyte complexes. Plasma levels of soluble P-selectin are significantly elevated in periodontitis and correlate with severity of disease and bacterial infection. Stimulation of endothelial cells with periodontopathogens results in rapid surface expression of P-selectin but does not induce NFκB translocation and subsequent de novo synthesis of P-selectin, E-selectin or ICAM-1. In platelets, bacterial stimulation leads to surface expression of P-selectin and fosters the formation of platelet-leukocyte aggregates within minutes. P-selectin is rapidly shed from the surface of platelets and endothelial cells and results in increased levels of soluble P-selectin. Periodontopathogens are able to directly cause activation of endothelial cells and platelets within minutes. Given that transient periodontitis-associated bacteremia commonly occurs after tooth brushing or chewing, our data suggest that reduction of periodontopathogens might result in potential cardiovascular benefits.
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  Contamination with recombinant IFN accounts for the unexpected stimulatory properties of commonly used IFN-blocking antibodies.

  Herwig P Moll, Harald Freudenthaler, Anna Zommer, Elisabeth Buchberger, Xiao-Hong Lin, Sara Crisafulli, Yognandan Pandya, Sidney Pestka, Thomas Lavoie, Christine Brostjan

  European journal of immunology. 01/2011; 41(1):252-4.
• 2.03

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  Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts.

  Patrick Starlinger, Lejla Alidzanovic, Dominic Schauer, Philipp Brugger, Silvia Sommerfeldt, Irene Kuehrer, Sebastian F Schoppmann, Michael Gnant, Christine Brostjan

  Disease markers. 01/2011; 31(2):55-65.

  The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. Variant... [more] The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated. Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF-4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P=0.006; P< 0.001). The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.
• 3.12

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  The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types.

  Herwig P Moll, Thomas Maier, Anna Zommer, Thomas Lavoie, Christine Brostjan

  Cytokine. 10/2010; 53(1):52-9.

  IFN-α proteins have been described to originate from 14 individual genes and allelic variants. However, the exceptional diversity of IFN-α and its functional impact are still poorly understood. To characterize the biological activity of IFN-α subtypes in relation to the cellular background, we inves... [more] IFN-α proteins have been described to originate from 14 individual genes and allelic variants. However, the exceptional diversity of IFN-α and its functional impact are still poorly understood. To characterize the biological activity of IFN-α subtypes in relation to the cellular background, we investigated the effect of IFN-α treatment in primary fibroblasts and endothelial cells of vascular or lymphatic origin. The cellular response was evaluated for 13 distinct IFN-α proteins with respect to transcript regulation of the IFN-stimulated genes (ISGs) IFIT1, ISG15, CXCL10, CXCL11 and CCL8. The IFN-α proteins displayed a remarkably consistent potency in gene induction irrespective of target gene and cellular background which led to the classification of IFN-α subtypes with low (IFN-α1), intermediate (IFN-α2a, -4a, -4b, -5, -16, -21) and high (IFN-α2b, -6, -7, -8, -10, -14) activity. The differential potency of IFN-α classes was confirmed at the ISG protein level and the functional protection of cells against influenza virus infection. Differences in IFN activity were only observed at subsaturating levels of IFN-α proteins and did not affect the time course of ISG regulation. Cell-type specific responses were apparent for distinct target genes independent of IFN-α subtype and were based on different levels of basal versus inducible gene expression. While fibroblasts presented with a high constitutive level of IFIT1, the expression in endothelial cells was strongly induced by IFN-α. In contrast, CXCL10 and CXCL11 transcript levels were generally higher in endothelial cells despite a pronounced induction by IFN-α in fibroblasts. In summary, the divergent potency of IFN-α proteins and the cell-type specific regulation of individual IFN target genes may allow for the fine tuning of cellular responses to pathogen defense.
• 6.07

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  Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.

  P Starlinger, H P Moll, A Assinger, C Nemeth, K Hoetzenecker, B Gruenberger, T Gruenberger, I Kuehrer, S F Schoppmann, M Gnant, C Brostjan

  Journal of thrombosis and haemostasis : JTH. 08/2010; 8(8):1809-19.

  Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis. Protein isoforms of platelet-derived thrombospondin-1... [more] Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis. Protein isoforms of platelet-derived thrombospondin-1 (TSP-1) were investigated for their potential to identify in vitro platelet activation when monitoring in vivo processes. TSP-1 was determined in plasma, serum or supernatant of purified platelets by ELISA and immunoblotting and was compared with standard markers of platelet activation. A collective of 20 healthy individuals and 30 cancer patients was analyzed.  While in vitro platelet degranulation led to a selective increase in the 200-kDa full-length molecule, an in vivo process involving platelet activation such as wound healing resulted in the predominant rise of the 140-kDa TSP-1 protein. The physiological ratio of circulating TSP-1 variants was determined and a cut-off level at 1.0 was defined to identify plasma samples with artificial in vitro platelet activation exceeding the cut-off level. In contrast, cancer patients known to frequently exhibit increased in vivo activation of platelets presented with a significantly decreased ratio of TSP-1 variants as compared with healthy volunteers. In comparison to standard platelet markers, TSP-1 constitutes a sensitive and stable parameter suited to monitor in vitro platelet activation. The analysis of TSP-1 protein isoforms further offers a valuable tool to reliably discriminate between in vitro and in vivo effects, to exclude variability introduced during blood processing and improve clinical monitoring.
• 5.13

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  Identification of Non-HLA antigens targeted by alloreactive antibodies in patients undergoing chronic hemodialysis.

  Senada Bilalic, Michael Veitinger, Karl-Heinz Ahrer, Viktoria Gruber, Maria Zellner, Christine Brostjan, Gregor Bartel, Daniel Cejka, Christian Reichel, Veronika Jordan, Christopher Burghuber, Ferdinand Mühlbacher, Georg A Böhmig, Rudolf Oehler

  Journal of proteome research. 02/2010; 9(2):1041-9.

  The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non... [more] The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non-HLA antigens in the serum of the recipient prior transplantation is associated with higher incidence of chronic rejection. However, the molecular identity of these antigens is largely unknown. To determine the most common non-HLA antigens, we tested lymphocytic extracts from 20 healthy volunteers with sera of 28 patients on the transplantation waiting list by Western blotting. There was a group of five proteins that was recognized by most sera. Using patient's own lymphocytes revealed that autoimmunity plays a minor role in this recognition. Two-dimensional Western blotting experiments followed by mass spectrometry identified the antigens as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. A detailed analysis of vimentin expression revealed that the antigenic 60 kDa isoform is underrepresented in patient's lymphocytes in comparison to those of healthy volunteers. The study revealed that preformed alloreactive antibodies are directed against a small number of specific protein isoforms. Our findings could provide a basis for future improvement of donor-recipient matching.
• 1.59

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  Pilot trial of autologous dendritic cells loaded with tumor lysate(s) from allogeneic tumor cell lines in patients with metastatic medullary thyroid carcinoma.

  Thomas Bachleitner-Hofmann, Josef Friedl, Michaela Hassler, Hubert Hayden, Peter Dubsky, Monika Sachet, Erwin Rieder, Roswitha Pfragner, Christine Brostjan, Stefan Riss, Bruno Niederle, Michael Gnant, Anton Stift

  Oncology reports. 07/2009; 21(6):1585-92.

  Immunotherapy with autologous dendritic cells (DCs) loaded with tumor lysate(s) from allogeneic tumor cell lines is a novel strategy to induce immune responses in cancer patients. We report on a pilot trial of autologous DCs pulsed with tumor cell lysate derived from allogeneic medullary thyroid car... [more] Immunotherapy with autologous dendritic cells (DCs) loaded with tumor lysate(s) from allogeneic tumor cell lines is a novel strategy to induce immune responses in cancer patients. We report on a pilot trial of autologous DCs pulsed with tumor cell lysate derived from allogeneic medullary thyroid carcinoma (MTC) cell lines in patients with metastatic MTC. The purpose of this study was to assess the safety, resulting immune responses and clinical activity of the DCs. DCs were injected into a groin lymph node at 3-week intervals. Monitoring included serial calcitonin tumor marker measurements, radiological imaging and immunological in vitro tests (T-cell Interferon-gamma detection assay, T-cell cytotoxicity assay). Ten patients (median age 47 years, range 29-77) were enrolled. DC vaccinations were well-tolerated and safe. After a median follow-up of 11 months, (range 7-26), 3 (30%) of 10 patients had stable disease, while 7 (70%) of the patients progressed during treatment. In 2 patients with stable disease, calcitonin decreased below treatment levels, paralleled by a T-cell-mediated immune response. Notably, treatment with DCs pulsed with a combination of different tumor cell lysates was followed by a calcitonin decrease in 4 patients who had previously experienced a calcitonin increase during monotherapy with DCs pulsed with a single lysate. Allogeneic tumor cell lysate-based DC immunotherapy is well-tolerated and safe. Combined treatment with different tumor cell lysate-pulsed DCs increases the likelihood of a calcitonin tumor marker response and should therefore be preferred over monotherapy with DCs pulsed with a single lysate.
• 2.50

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  Elevated levels of circulating endothelial progenitor cells in head and neck cancer patients.

  Markus Brunner, Dietmar Thurnher, Gregor Heiduschka, Matthaeus Ch Grasl, Christine Brostjan, Boban M Erovic

  Journal of surgical oncology. 10/2008;

  BACKGROUND AND OBJECTIVES: Measurement of circulating endothelial cells (CECs) and progenitor cells (EPCs) has potential as a surrogate marker for monitoring anticancer treatment. This study evaluated the significance of CECs and EPCs in the blood of patients with head and neck squamous cell carcino... [more] BACKGROUND AND OBJECTIVES: Measurement of circulating endothelial cells (CECs) and progenitor cells (EPCs) has potential as a surrogate marker for monitoring anticancer treatment. This study evaluated the significance of CECs and EPCs in the blood of patients with head and neck squamous cell carcinoma. METHODS: In a prospective trial fresh blood samples from 22 tumor patients and 18 controls were tested using multiparametric flow-cytometry. CECs were defined as CD31(+)/CD146(+) and CD45(-)/7AAD(-). EPCs were defined as CD133(+)/KDR(+) and CD3(-)/CD19(-)/CD33(-)/7AAD(-). RESULTS: Median levels (min/max) of CECs in the tumor group were 2 (0/5) at the time of diagnosis, 1 (0/5) 1 year after therapy and 2 (0/6) in the control cohort. Median levels of EPCs were 5 (1/41) before and 10 (0/21) after treatment in the tumor group compared to 2 (0/7) in the control cohort (P < 0.001 and P = 0.03). CEC and EPC levels showed no apparent correlation with tumor size and response to radiotherapy after 18 months of observation. CONCLUSIONS: In this pilot study CD133(+)/KDR(+) EPCs were significantly elevated in head and neck tumor patients before and after therapy. Our results warrant further studies on the use of EPCs as a surrogate marker for anticancer therapies in these patients. J. Surg. Oncol. (c) 2008 Wiley-Liss, Inc.
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  Neoadjuvant treatment of colorectal cancer with bevacizumab: the perioperative angiogenic balance is sensitive to systemic thrombospondin-1 levels.

  Christine Brostjan, Kristina Gebhardt, Birgit Gruenberger, Verena Steinrueck, Halina Zommer, Harald Freudenthaler, Sebastian Roka, Thomas Gruenberger

  Clinical cancer research : an official journal of the American Association for Cancer Research. 05/2008; 14(7):2065-74.

  PURPOSE: Colorectal cancer patients receiving neoadjuvant treatment with bevacizumab, a monoclonal antibody neutralizing vascular endothelial growth factor (VEGF), may suffer from wound healing complications after surgery as the antibody persists in patient blood. We characterized the systemic angio... [more] PURPOSE: Colorectal cancer patients receiving neoadjuvant treatment with bevacizumab, a monoclonal antibody neutralizing vascular endothelial growth factor (VEGF), may suffer from wound healing complications after surgery as the antibody persists in patient blood. We characterized the systemic angiogenic balance in the perioperative period to evaluate its effect on physiologic angiogenesis. EXPERIMENTAL DESIGN: Nineteen patients receiving combination chemotherapy and bevacizumab for six neoadjuvant cycles were compared with 14 patients receiving chemotherapy without bevacizumab. Plasma from perioperative days -1, +1, +7, and +21 was analyzed for VEGF, thrombospondin-1 (TSP-1), and PD-ECGF concentrations. The angiogenic capacity was further tested in an in vitro assay of endothelial cell proliferation and migration. RESULTS: On day +1, the onset of wound healing was reflected in a change of balance, i.e., an increase of proangiogenic factors VEGF and platelet-derived endothelial cell growth factor compared with low TSP-1 inhibitor levels in both treatment groups. Patients with bevacizumab therapy showed significantly higher blood levels of total VEGF throughout the evaluation period. However, most VEGF molecules were inactive, i.e., complexed with antibody. Nevertheless, the capacity to stimulate endothelial growth was higher for these plasma samples and was reflected in low TSP-1 levels and an altered TSP-1 sensitivity. When purified TSP-1 protein was added, plasma samples of the bevacizumab but not the chemotherapy group showed reduced endothelial growth. CONCLUSIONS: Feedback mechanisms of bevacizumab therapy are not restricted to VEGF expression but seem to involve additional factors, such as TSP-1, which influences the systemic angiogenic balance and permits endothelial growth.
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  Neutralizing type I IFN antibodies trigger an IFN-like response in endothelial cells.

  Herwig Peter Moll, Harald Freudenthaler, Anna Zommer, Elisabeth Buchberger, Christine Brostjan

  Journal of immunology (Baltimore, Md. : 1950). 05/2008; 180(8):5250-6.

  Neutralizing Abs to type I IFNs are of therapeutic significance, i.e., are currently evaluated for the treatment of autoimmune diseases with pathogenic IFN-alpha production such as for systemic lupus erythematosus. Unexpectedly, we observed that several neutralizing Abs reportedly known to counterac... [more] Neutralizing Abs to type I IFNs are of therapeutic significance, i.e., are currently evaluated for the treatment of autoimmune diseases with pathogenic IFN-alpha production such as for systemic lupus erythematosus. Unexpectedly, we observed that several neutralizing Abs reportedly known to counteract IFN-alpha or IFN-beta activity triggered an "IFN-like" response in quiescent primary human endothelial cells leading to activation of the transcription factor IFN-stimulated gene factor 3 and the expression of IFN-responsive genes. Furthermore, these Abs were found to enhance rather than inhibit type I IFN signals, and the effect was also detectable for distinct other cell types such as PBMCs. The stimulatory capacity of anti-IFN-alpha/beta Abs was mediated by the constitutive autocrine production of "subthreshold" IFN levels, involved the type I IFNR and was dependent on the Fc Ab domain, as Fab or F(ab')(2) fragments potently inhibited IFN activity. We thus propose that a combined effect of IFN recognition by the Ab paratope and the concomitant engagement of the Fc domain may trigger an IFN signal via the respective type I IFNR, which accounts for the observed IFN-like response to the neutralizing Abs. With respect to clinical applications, the finding may be of importance for the design of recombinant Abs vs Fab or F(ab')(2) fragments to efficiently counteract IFN activity without undesirable activating effects.
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  Effects of 15d-PGJ(2) on VEGF-induced angiogenic activities and expression of VEGF receptors in endothelial cells.

  Philip Funovics, Christine Brostjan, Anneliese Nigisch, Anna Fila, Anna Grochot, Katarzyna Mleczko, Halina Was, Guenter Weigel, Jozef Dulak, Alicja Jozkowicz

  Prostaglandins & other lipid mediators. 06/2006; 79(3-4):230-44.

  15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) upregulates expression of vascular endothelial growth factor (VEGF), but may inhibit angiogenesis. We found that 15d-PGJ(2) (1-10muM) attenuated all VEGF-induced angiogenic activities in human umbilical vein endothelial cells (HUVEC). It blocked ... [more] 15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) upregulates expression of vascular endothelial growth factor (VEGF), but may inhibit angiogenesis. We found that 15d-PGJ(2) (1-10muM) attenuated all VEGF-induced angiogenic activities in human umbilical vein endothelial cells (HUVEC). It blocked almost completely cell proliferation, potently reduced migration, assembly into tube-like network on matrigel, and growth of capillaries into collagen gel. 15d-PGJ(2) inhibited expression of VEGFR-1 and VEGFR-2 receptors both at mRNA and protein levels. This inhibition, however, was transient (observed after 6-12h, but not after 24h) and weak (20-30%), and could not fully explain inhibition of response to VEGF. Accordingly, proliferation was inhibited when 15d-PGJ(2) was added 24h after VEGF or in cells stimulated with basic fibroblast growth factor. Interestingly, 15d-PGJ(2) decreased activities of c-jun and c-myc in HUVEC and overexpression of c-myc attenuated its antiproliferative effects. This suggests that inhibition of this transcription factor by 15d-PGJ(2) contributes to decrease in angiogenic response.
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  Retroviral targeting of proliferating endothelial cells.

  Alexander Gornikiewicz, Anna Zommer, Raimund Jakesz, Michael Gnant, Christine Brostjan

  Acta biochimica Polonica. 02/2005; 52(3):731-5.

  Tumor growth requires the formation of new blood vessels by endothelial cells. Thus, surface molecules -- such as angiogenin receptors -- that are selectively expressed on growing endothelium represent an attractive target for directed delivery of compounds to tumor tissue. We attempted to obtain ge... [more] Tumor growth requires the formation of new blood vessels by endothelial cells. Thus, surface molecules -- such as angiogenin receptors -- that are selectively expressed on growing endothelium represent an attractive target for directed delivery of compounds to tumor tissue. We attempted to obtain genetically engineered retroviral vectors targeted to the endothelium by inserting the human angiogenin sequence into Moloney murine leukemia virus envelope glycoprotein. Abundant expression of the chimeric protein could be verified. However, while being selective for proliferating human endothelial cells, the recombinant retroviral particles displayed low transduction efficiencies and thus have to be further improved.
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  Dendritic cell vaccination in medullary thyroid carcinoma.

  Anton Stift, Monika Sachet, Rubina Yagubian, Clemens Bittermann, Peter Dubsky, Christine Brostjan, Roswitha Pfragner, Bruno Niederle, Raimund Jakesz, Michael Gnant, Josef Friedl

  Clinical cancer research : an official journal of the American Association for Cancer Research. 06/2004; 10(9):2944-53.

  PURPOSE: Prognosis and treatment effectiveness for medullary thyroid carcinoma (MTC) are strictly related to tumor stage. Palliative treatment options show no significant benefit. A promising treatment approach for human cancer is based on the vaccination of autologous dendritic cells (DCs). EXPERIM... [more] PURPOSE: Prognosis and treatment effectiveness for medullary thyroid carcinoma (MTC) are strictly related to tumor stage. Palliative treatment options show no significant benefit. A promising treatment approach for human cancer is based on the vaccination of autologous dendritic cells (DCs). EXPERIMENTAL DESIGN: The objective of this study was to evaluate the effectiveness of DC vaccines in MTC patients. Therefore, we generated autologous tumor lysate-pulsed DCs from 10 patients suffering from advanced MTC for repeated vaccination. Mature DCs were derived from peripheral blood monocytes by using CD14 magnetic bead selection and subsequent culture in the presence of granulocyte macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha with or without addition of IFN-gamma. DCs were loaded with tumor lysate and further injected into a groin lymph node. Toxicity, tumor marker profile, immune response, and clinical response were determined. RESULTS: Vaccination was well tolerated and induced a positive immunological response in all of the tested patients as evaluated by in vivo delayed-type hypersensitivity reactivity or in vitro intracytoplasmic IFN-gamma detection assay. Three patients had a partial response, 1 patient presented a minor response, and 2 patients showed stable disease. The remaining 4 patients had progressive disease. CONCLUSIONS: These data provide strong evidence that vaccination with tumor-lysate pulsed DCs results in the induction of a specific immune response in patients suffering from MTC. Objective clinical responses could be observed even for far-advanced disease. Therefore, we suggest that MTC is particularly suited for DC-based immunotherapy.
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  Monitoring of circulating angiogenic factors in dendritic cell-based cancer immunotherapy.

  Christine Brostjan, Anita Bayer, Anna Zommer, Alexander Gornikiewicz, Sebastian Roka, Thomas Benkö, Rubina Yaghubian, Raimund Jakesz, Günther Steger, Michael Gnant, Josef Friedl, Anton Stift

  Cancer. 12/2003; 98(10):2291-301.

  BACKGROUND: A promising treatment approach for patients with malignant disease that recently has found its way into clinical trials is based on vaccination with autologous dendritic cells loaded with tumor antigens. However, adequate assays for monitoring clinical and immunologic responses still are... [more] BACKGROUND: A promising treatment approach for patients with malignant disease that recently has found its way into clinical trials is based on vaccination with autologous dendritic cells loaded with tumor antigens. However, adequate assays for monitoring clinical and immunologic responses still are under debate. In recent years, the determination of angiogenic markers has shown considerable potential in the diagnosis and prognosis of patients with malignant disease, because tumor growth and spread are promoted by angiogenesis, the formation of new blood vessels. METHODS: The authors established a method for measuring the plasma levels of three modulators of angiogenesis: vascular endothelial growth factor, platelet-derived endothelial cell growth factor, and thrombospondin-1. The angiogenic blood profile of a healthy control group was characterized and compared with a group of patients with malignant disease. Ultimately, levels of circulating angiogenic factors were monitored in the course of dendritic cell-based cancer immunotherapy. RESULTS: Baseline levels of angiogenic mediators varied substantially among healthy individuals but showed consistent values for each individual. Blood levels of circulating angiogenic factors were elevated significantly in patients with advanced disease and were highly sensitive to dendritic cell-based immunotherapy. CONCLUSIONS: To our knowledge, the current report was the first to analyze circulating levels of angiogenic factors during dendritic cell-based immunotherapy. The authors observed a noteworthy change in the angiogenic blood profile with treatment, and this change was correlated with the induction of an immunologic response.
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  Glutamine starvation of monocytes inhibits the ubiquitin-proteasome proteolytic pathway.

  Maria Zellner, Christopher Gerner, Maja Munk Eliasen, Susanne Wurm, Jürgen Pollheimer, Andreas Spittler, Christine Brostjan, Erich Roth, Rudolf Oehler

  Biochimica et biophysica acta. 08/2003; 1638(2):138-48.

  Peripheral blood monocytes utilize free glutamine (Gln) in addition to glucose as an important energy substrate. Although this demand increases upon activation, monocytes are commonly confronted with decreased plasma Gln during critical illness and thus suffer from Gln-starvation. Here we investigat... [more] Peripheral blood monocytes utilize free glutamine (Gln) in addition to glucose as an important energy substrate. Although this demand increases upon activation, monocytes are commonly confronted with decreased plasma Gln during critical illness and thus suffer from Gln-starvation. Here we investigate the influence of Gln-starvation on protein stability and its effects on the monocyte proteome. Gln-starvation caused a reduction of protein degradation which was accompanied by an accumulation of ubiquitin-protein conjugates and a reduction of intracellular ATP. Similar effects were observed under ATP-reducing conditions and in the presence of a proteasome inhibitor. Using two-dimensional gel electrophoresis we identified the IL-1beta precursor protein (pIL-1beta) as the, by far, most induced protein in endotoxin-treated monocytes. The degradation of the short-lived pIL-1beta was strongly reduced during Gln-starvation, while the degradation of the long-lived, constitutively expressed beta-actin was less affected. This indicates that although Gln-starvation reduces protein breakdown on the overall proteasome level, it leads to differential changes in the stability of specific proteins. This selective effect is likely to contribute to the immunocompromised state of monocytes commonly observed during critical illness.
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  In vivo induction of dendritic cell-mediated cytotoxicity against allogeneic pancreatic carcinoma cells.

  Anton Stift, Josef Friedl, Peter Dubsky, Thomas Bachleitner-Hofmann, Thomas Benkoe, Christine Brostjan, Raimund Jakesz, Michael Gnant

  International journal of oncology. 04/2003; 22(3):651-6.

  The objective of this study was to assess the toxicity and immunological response induced by autologous dendritic cells (DCs) pulsed with allogeneic tumor lysate in a pancreatic cancer patient. The lack of available tumor peptides in pancreatic cancer strongly supports the idea to use allogeneic tum... [more] The objective of this study was to assess the toxicity and immunological response induced by autologous dendritic cells (DCs) pulsed with allogeneic tumor lysate in a pancreatic cancer patient. The lack of available tumor peptides in pancreatic cancer strongly supports the idea to use allogeneic tumor cells as a source of antigens. The patient suffering from a stage IV pancreatic cancer received 1-2x10(7) autologous monocyte-derived DCs in three-week intervals injected into a groin lymph node. Monocytes from peripheral blood were isolated by magnetic bead selection. For the first ten vaccinations DCs were loaded with autologous tumor cell lysate obtained during surgical exploration. After consumption of the autologous lysate, equal numbers of DCs were pulsed with lysate of the tumor cell line AsPc-1 and BxPc-3 for a further five vaccinations. Peripheral mononuclear cells (PMNCs) were harvested after the seventh and compared with PMNCs obtained after the fourteenth vaccination for immunological response. Delayed type hypersensitivity reactivity to DCs pulsed with autologous and allogeneic tumor lysate was also assessed. The patient received a total of fifteen vaccinations. There was no toxicity or evidence of autoimmunity observed. Delayed type hypersensitivity was found to be positive for the autologous as well as the allogeneic tumor lysate pulsed DCs. in vitro cytotoxicity assays demonstrated a dramatic increase of the PMNC killing capacity against the pancreatic cancer cell lines AsPc-1 and BxPc-3 after the fourteenth compared to the seventh vaccination. CT scans revealed a stable disease for six months. The administration of autologous DCs pulsed with allogeneic tumor lysate is non-toxic and suitable for inducing an immunological anti-tumor response. Even though this study was confined to a single patient, the data might open a door for novel immunotherapeutical strategies.
• 17.79

  Impact points

  Dendritic cell-based vaccination in solid cancer.

  A Stift, J Friedl, P Dubsky, T Bachleitner-Hofmann, G Schueller, T Zontsich, T Benkoe, K Radelbauer, C Brostjan, R Jakesz, M Gnant

  Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 02/2003; 21(1):135-42.

  PURPOSE: Dendritic cell (DC)-based immunotherapy is rapidly emerging as a viable tool in cancer treatment. This approach has been used mostly in patients in the presence of defined tumor antigens such as melanoma. In this study, cancer patients with advanced disease that lacks defined tumor antigens... [more] PURPOSE: Dendritic cell (DC)-based immunotherapy is rapidly emerging as a viable tool in cancer treatment. This approach has been used mostly in patients in the presence of defined tumor antigens such as melanoma. In this study, cancer patients with advanced disease that lacks defined tumor antigens were vaccinated with tumor lysate-pulsed DCs. PATIENTS AND METHODS: Twenty patients (pancreatic, hepatocellular, cholangiocellular, and medullary thyroid carcinoma) with stage IV disease were enrolled in the study. In 3-week intervals, freshly isolated autologous CD14 magnetic bead-selected monocytes were cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to obtain immature DCs. These cells were pulsed with autologous tumor lysate and matured with tumor necrosis factor alpha. Mature DCs were applied into a groin lymph node, under ultrasound guidance. Adjuvant interleukin-2 (20,000 U/kg) was given subcutaneously daily, for 12 days, after each vaccination. Toxicity, tumor marker profile, immune response, and clinical response were determined. RESULTS: Vaccination was well tolerated. No physical signs of autoimmunity were detected. DC vaccination induced delayed-type hypersensitivity reactivity in 18 patients. Tumor marker responses were observed in eight patients. In addition, in three patients the generation of interferon gamma-positive T cells was induced during the vaccination. Objective changes in measurable lesions or tumor markers were evident in seven of 20 assessed patients. None of the patients was found to meet the criteria for partial or complete responses. CONCLUSION: These data indicate that vaccination with autologous tumor-pulsed DCs generated from peripheral blood is safe and can induce tumor-specific cellular cytotoxicity. Clinical responses are achievable, even in patients with advanced disease.
• 2.35

  Impact points

  Differential expression of inhibitory and activating CD94/NKG2 receptors on NK cell clones.

  Christine Brostjan, Teresa Bellón, Yuri Sobanov, Miguel López-Botet, Erhard Hofer

  Journal of immunological methods. 07/2002; 264(1-2):109-19.

  Natural killer cells are known to express a variety of surface receptors involved in HLA class I monitoring. It is thus of interest to investigate the clonal distribution and relative expression levels of activating versus inhibitory NK receptors. We have developed a quantitative real-time reverse t... [more] Natural killer cells are known to express a variety of surface receptors involved in HLA class I monitoring. It is thus of interest to investigate the clonal distribution and relative expression levels of activating versus inhibitory NK receptors. We have developed a quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay designed to determine specific and absolute mRNA levels for NKG2-A/B, -C, -E, -F, -H and NKG2-D. When analyzing NK cell clones derived from a single donor we found differential expression of inhibitory (NKG2-A/B) versus triggering (NKG2-C and potentially -E, -F, -H) NK receptor chains. The generation of the splice variants NKG2-E and -H seemed to occur at a constant ratio. We further compared NKG2 transcript levels to surface receptor expression as monitored by flow cytometric analysis and to NK cell cytotoxicity as detected by reverse ADCC: a clear correlation was observed. Thus, the data obtained reveal a substantial variability in the NKG2 repertoire among NK cell subpopulations, which is likely to affect the sensitivity and reactivity towards the ligand HLA-E.